: The aim of this study was to investigate the occurrence, diversity, and distribution of Phytophthora species in Protected Natural Areas (PNAs), including forest stands, rivers, and riparian ecosystems, in Sicily (Italy), and assessing correlations with natural vegetation and host plants. Fifteen forest stands and 14 rivers in 10 Sicilian PNAs were studied. Phytophthora isolations from soil and stream water were performed using leaf baitings. Isolates were identified using both morphological characters and sequence analysis of the internal transcribed spacer (ITS) region. A rich community of 20 Phytophthora species from eight phylogenetic clades, including three new Phytophthora taxa, was recovered (17 species in rhizosphere soil from forest stands and 12 species in rivers). New knowledge about the distribution, host associations, and ecology of several Phytophthora species was provided.